Joint Destruction Is Associated With All Types of Cardiovascular Events in French Rheumatoid Patients: A Real-Life Study With Very Long Follow-Up.

Objective: Rheumatoid arthritis (RA) leads not only to joint destruction but also to systemic manifestations, with an increased incidence of cardiovascular events (CVE). Many studies have shown a link between RA severity and CV risk, but the duration of follow-up remains often insufficient to allow a conclusion. The CVE definition was generally reduced to myocardial infarction and stroke, and few studies were conducted in non-Anglo-Saxon countries with low CV incidence. This study aimed to assess the relationship between joint destruction and the occurrence of different types of CVE in a large cohort of French RA patients with a long-term follow-up. Methods: This historical cohort study included 571 RA patients followed between 1992 and 2012 in Lyon, France. The primary endpoint was the first occurrence of a CVE. Logistic regressions were used to identify factors associated with CVE occurrence. Cox proportional hazard models were performed as a separate analysis to take advantage of the long-term follow-up. Results: During a mean follow-up of 16.1 years, 30.3% of patients experienced a CVE, mostly acute arterial events. Joint destruction was associated with an increased risk of CVE [odds ratio = 3.72; 95% confidence interval (CI), 1.09-15.35; p = 0.047] among non-smoker RA patients. A survival analysis revealed that joint destruction was associated with a shorter time to onset of the first CVE only among non-smokers (hazard ratio = 3.44; 95% CI, 1.07-11.04; p = 0.038). Conclusion: Joint destruction is associated with CVE occurrence in RA patients from a population with a lower incidence of CV disease. This study suggests that RA patients, especially those with destruction, merit the institution of precise guidelines to manage this CV risk, and trials are required to evaluate them.

Synergistic Interaction Between High Bioactive IL-17A and Joint Destruction for the Occurrence of Cardiovascular Events in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) remains a cause of morbidity and mortality in many patients while new treatments have changed the face of the disease. Despite the emergence of these new drugs, cardiovascular (CV) diseases remain more frequent in RA patients compared with the general population. However, predictive biomarkers of RA severity and precise guidelines to manage the CV risk in these patients are still lacking. Pro-inflammatory cytokines contribute both to RA and CV pathogenesis. Focusing on IL-17A, high levels of bioactive IL-17A were associated with destruction in RA but also during myocardial infarction. The study aimed to assess the relationship between bioactive IL-17A, destruction and the occurrence of CV events (CVE) in RA patients with a very long follow-up. Thirty-six RA patients were followed between 1970 and 2012 in Lyon, France. They were tested for bioactive IL-17A and clinical and biological characteristics were recorded at baseline. Then, the occurrence of CVE was registered during the follow-up. To study the bioactive fraction of IL-17A, the bioassay used the ability of human umbilical vein endothelial cells to produce IL-8 in presence of RA plasma samples with or without an anti-IL-17A antibody. Bioactive IL-17A level at baseline was higher in RA patients who later experienced a CVE compared to those without (0.77 vs 0.21 ng/ml, p-value = 0.0095, Mann-Whitney test) and synergized with joint destruction (p-value = 0.020, Kruskal-Wallis test). Through its effects on vessels and thrombosis, high levels of bioactive IL-17A could represent a long-term marker of CV risk.

Interleukin-25 Produced by Synoviocytes Has Anti-inflammatory Effects by Acting As a Receptor Antagonist for Interleukin-17A Function.

The production and function of cytokines are highly regulated. One mechanism is the balance between pro- and anti-inflammatory cytokines. As interleukin (IL)-17A and IL-25 share the IL-17RA receptor chain, we hypothesize that IL-25 acts as an IL-17A receptor antagonist and limits its pro-inflammatory effects. The production and expression kinetics of IL-25 and its receptor chains IL-17RA and RB were analyzed in rheumatoid synoviocytes alone or in coculture with peripheral blood mononuclear cells (PBMCs). The effects of autocrine or exogenous IL-25 on synoviocytes were investigated in the presence or not of an anti-IL-25 antibody. To study the regulatory effects of IL-25, synoviocytes and/or PBMCs were exposed to IL-25 before being treated with IL-17A and tumor necrosis factor alpha (TNF-α) alone or combined. IL-25, IL-6, and bioactive IL-17A were quantified in rheumatoid arthritis (RA) patient plasma. Synoviocytes expressed and secreted IL-25, and expressed the two chains of its receptor IL-17RA and IL-17RB. IL-17RB expression was increased by TNF-α. IL-25 production occurred at a delayed time point (5 days) after stimulation with IL-17A and TNF-α. Synoviocytes pretreated with IL-25 were less responsive to IL-17A and TNF-α. PBMCs exposed to IL-25 showed a decreased production of pro-inflammatory mediators, including IL-17A with a 57% decrease; p = 0.002. IL-25 levels were elevated in the plasma of RA patients compared to healthy subjects (p = 0.03). However, these levels are not high enough to inhibit the function of circulating IL-17A. In conclusion, it was shown for the first time that synoviocytes produce IL-25, specifically at late time points and that IL-25 acts as a regulator of IL-17A-driven inflammation, as indicated by in vitro results and in vivo, in a long-term RA patient follow-up. These results may be important when considering IL-17A inhibition.

Evaluation of Anti-inflammatory Effects of Steroids and Arthritis-Related Biotherapies in an In Vitro Coculture Model with Immune Cells and Synoviocytes.

BACKGROUND: During rheumatoid arthritis (RA), steroids and biotherapies are used alone and combined. Efficacy has been established in clinical trials but their differential effects at the cellular level are less documented. The aim was to study these cellular effects using an in vitro model with synoviocytes interacting with peripheral blood mononuclear cells (PBMC) to reproduce the interactions in the RA synovium. METHODS: Activated-PBMC were cocultured with RA synoviocytes during 48 h. A dose-response of methylprednisolone (MP) was tested and different biotherapies (Infliximab, Etanercept, Adalimumab, Tocilizumab, Abatacept, and Rituximab) were added alone or in combination with MP. Cytokine production (IL-17, IL-6, IL-1ß, IFN-γ and IL-10) was measured by ELISA. RESULTS: Addition of MP to cocultures inhibited the production of all cytokines. The response to the biotherapies alone was treatment-dependent. IL-17 production was inhibited only by Tocilizumab (p = 0.004), while IL-6 was decreased only by Infliximab (p ≤ 0.002). IL-1ß level was affected in all conditions (p ≤ 0.03). IFN-γ production was mainly decreased by Infliximab (p = 0.004) and IL-10 by Infliximab and Tocilizumab (p ≤ 0.004). The combination MP and biotherapies did not induce an additional effect on pro-inflammatory cytokine inhibition. The combination MP and biotherapies induced a higher IL-10 secretion than MP alone, mainly with Rituximab. CONCLUSION: Steroids inhibited the secretion of all cytokines, and low doses were as potent. The anti-inflammatory effect of biotherapies was dependent on their mechanism of action. MP and biotherapy combination did not enhance the inhibitory effect on pro-inflammatory cytokines but could have a beneficial effect by increasing IL-10 production.

Interaction among activated lymphocytes and mesenchymal cells through podoplanin is critical for a high IL-17 secretion.

BACKGROUND: During chronic inflammation, immune cells, notably Th17 cells, infiltrate the inflammatory site and interact with local mesenchymal cells. Applied to rheumatoid arthritis (RA), the aim is to study the interactions between synoviocytes and peripheral blood mononuclear cells (PBMC) with a focus on the Th17 pathway and to identify a mechanism which leads to high IL-17 secretion with an interest on podoplanin. METHODS: PBMC from healthy donors and RA patients were co-cultured with RA synoviocytes during 48 h, in the presence or not of phytohemagglutinin. An antibody against podoplanin was used in co-culture. Cytokine production (IL-6, IL-1ß and IL-17) was measured by ELISA and cell staining (CD3, CD4, IL-17 and podoplanin) by flow cytometry. RESULTS: In control conditions, IL-6 and IL-1ß production was increased in PBMC-synoviocyte co-culture compared to PBMC alone (p = 0.02). No additional effect was observed with PBMC activation. Flow cytometry analysis showed no difference in the percentage of Th17 cells in activated PBMC alone or with synoviocytes (p = 0.4), indicating that Th17 differentiation requires only T cell activation. Conversely, IL-17 production was highly increased in co-cultures with activated PBMC vs. activated PBMC alone (p = 0.002). Transwell experiments confirm that cell-cell contact was critical for IL-17 secretion. The incubation of either PBMC or synoviocytes with an anti-podoplanin antibody decreased IL-17 secretion by 60 % (p = 0.008). CONCLUSIONS: Interactions between resting PBMC and synoviocytes are sufficient to induce IL-6 and IL-1ß production. Both PBMC activation and cell interactions are needed to induce a high IL-17 secretion. Podoplanin contributes at the level of both lymphocytes and synoviocytes.

Differential Effects of IL-17A and TNF-α on Osteoblastic Differentiation of Isolated Synoviocytes and on Bone Explants from Arthritis Patients.

OBJECTIVE: TNF-α and IL-17A act on fibroblast-like synoviocytes (FLS) and contribute to cytokine production, inflammation, and tissue destruction in rheumatoid arthritis (RA). The aim of this study was to compare their effects on osteogenic differentiation of isolated FLS and on whole bone explants from RA and osteoarthritis (OA) patients. METHODS: Fibroblast-like synoviocytes and bone explants were cultured in the presence or absence of TNF-α and/or IL-17A. Mineralization of extracellular matrix of FLS was measured by alizarin red and alkaline phosphatase activity (ALP). mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2, and RUNX2, key genes associated with osteogenesis. IL-6 and IL-8 levels were measured by enzyme-linked immunosorbent assays. Bone explant structure was quantified by histomorphometry. RESULTS: In isolated OA and RA FLS, the combination of TNF-α and IL-17A induced matrix mineralization, increased ALP activity and expression of the osteogenesis-associated genes Wnt5a, BMP2, and Runx2, indicating an osteogenic differentiation. Wnt5a levels increased with TNF-α alone and in combination with IL-17A. BMP2 expression decreased with IL-17A and TNF-α after 12 h with OA FLS and 24 h with RA FLS. Runx2 expression decreased only with combination of TNF-α and IL-17A in OA FLS and with cytokines alone and combined in RA FLS. IL-6 and IL-8 production increased with IL-17A and/or TNF-α in both FLS and bone samples, especially from RA. Treatment of bone explants with cytokine combination increased ALP in OA but not RA samples. A decrease in bone volume was seen with cytokine combination, especially with RA explants. CONCLUSION: Differences were observed for the effects of IL-17A and TNF-α on osteogenic differentiation. In isolated FLS, increased osteoblastogenesis was observed, contrasting with the inhibitory effect in whole bone, specifically in RA. The net effect of IL-17A and TNF-α appears to depend on the disease state and the presence of other cells.

IgM rheumatoid factor amplifies the inflammatory response of macrophages induced by the rheumatoid arthritis-specific immune complexes containing anticitrullinated protein antibodies.

OBJECTIVES: Anticitrullinated protein antibodies (ACPA) are specifically associated with rheumatoid arthritis (RA) and produced in inflamed synovial membranes where citrullinated fibrin, their antigenic target, is abundant. We showed that immune complexes containing IgG ACPA (ACPA-IC) induce FcγR-mediated tumour necrosis factor (TNF)-α secretion in macrophages. Since IgM rheumatoid factor (RF), an autoantibody directed to the Fc fragment of IgG, is also produced and concentrated in the rheumatoid synovial tissue, we evaluated its influence on macrophage stimulation by ACPA-IC. METHODS: With monocyte-derived macrophages from more than 40 healthy individuals and different human IgM cryoglobulins with RF activity, using a previously developed human in vitro model, we evaluated the effect of the incorporation of IgM RF into ACPA-IC. RESULTS: IgM RF induced an important amplification of the TNF-α secretion. This effect was not observed in monocytes and depended on an increase in the number of IgG-engaged FcγR. It extended to the secretion of interleukin (IL)-1ß and IL-6, was paralleled by IL-8 secretion and was not associated with overwhelming secretion of IL-10 or IL-1Ra. Moreover, the RF-induced increased proinflammatory bioactivity of the cytokine response to ACPA-IC was confirmed by an enhanced, not entirely TNF-dependent, capacity of the secreted cytokine cocktail to prompt IL-6 secretion by RA synoviocytes. CONCLUSIONS: By showing that it can greatly enhance the proinflammatory cytokine response induced in macrophages by the RA-specific ACPA-IC, these results highlight a previously undescribed, FcγR-dependent strong proinflammatory potential of IgM RF. They clarify the pathophysiological link between the presence of ACPA and IgM RF, and RA severity.

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes.

UNLABELLED: HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.